217 research outputs found
The development of signal transduction pathways during epididymal maturation is calcium dependent
No full textHeath Ecroyd, Kelly L. Asquith, Russell C. Jones and R. John Aitkenhttp://www.elsevier.com/wps/find/journaldescription.cws_home/622816/description#descriptio
Enhanced molecular chaperone activity of the small heat-shock protein alpha beta-crystallin following covalent immobilization onto a solid-phase support
No full textWe report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme−inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s−1 for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.Megan Garvey, Stefani S. Griesser, Hans J. Griesser, Benjamin Thierry, Matthew R. Nussio, Joseph G. Shapter, Heath Ecroyd, Sofia Giorgetti, Vittorio Bellotti, Juliet A. Gerrard and John A. Carve
AR-12 Inhibits Chaperone Proteins Preventing Virus Replication and the Accumulation of Toxic Misfolded Proteins
No full textFil: Booth, Laurence. Virginia Commonwealth University; Estados UnidosFil: Roberts, Jane L. Virginia Commonwealth University; Estados UnidosFil: Ecroyd, Heath. University of Wollongong; AustraliaFil: Reid, St Patrick. United States Army Medical Research Institute of Infectious Diseases; Estados UnidosFil: Proniuk, Stefan. Arno Therapeutics; Estados UnidosFil: Zukiwski, Alexander. Arno Therapeutics; Estados UnidosFil: Jacob, Abraham. University of Arizona; Estados UnidosFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Tunon, Maria J. Universidad de León; EspañaFil: Dent, Paul. Virginia Commonwealth University; Estados Unido
Avoiding the oligomeric state: AlphaB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils
No full textThe in vivo aggregation of proteins into amyloid fibrils suggests that cellular mechanisms that normally prevent or reverse this aggregation have failed. The small heat-shock molecular chaperone protein αB-crystallin (αB-c) inhibits amyloid formation and colocalizes with amyloid plaques; however, the physiological reason for this localization remains unexplored. Here, using apolipoprotein C-II (apoC-II) as a model fibril-forming system, we show that αB-c binds directly to mature amyloid fibrils (Kd 5.4 ± 0.5 μM). In doing so, αB-c stabilized the fibrils from dilution-induced fragmentation, halted elongation of partially formed fibrils, and promoted the dissociation of mature fibrils into soluble monomers. Moreover, in the absence of dilution, the association of αB-c with apoC-II fibrils induced a 14-fold increase in average aggregate size, resulting in large fibrillar tangles reminiscent of protein inclusions. We propose that the binding of αB-c to fibrils prevents fragmentation and mediates the lateral association of fibrils into large inclusions. We further postulate that transient interactions of apoC-II with αB-c induce a fibril-incompetent monomeric apoC-II form, preventing oligomerization and promoting fibril dissociation. This work reveals previously unrecognized mechanisms of αB-c chaperone action in amyloid assembly and fibril dynamics, and provides a rationale for the in vivo colocalization of small heat-shock proteins with amyloid deposits.—Binger, K. J., Ecroyd, H., Yang, S., Carver, J. A., Howlett, G. J., Griffin, M. D. W. Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils.Katrina J. Binger, Heath Ecroyd, Shuo Yang, John A. Carver, Geoffrey J. Howlett and Michael D. W. Griffi
Post-testicular sperm environment and fertility
No full textJ-L. Gatti, S. Castella, F. Dacheux, H. Ecroyd, S. Métayer, V. Thimon and J-L. Dacheuxhttp://www.elsevier.com/wps/find/journaldescription.cws_home/503300/description#descriptio
Unravelling the mysteries of protein folding and misfolding
No full textArticle first published online: 2 SEP 2008This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for example, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.Heath Ecroyd and John A. Carve
The effect of small molecules in modulating the chaperone activity of alphaB-crystallin against ordered and disordered protein aggregation
No full textProtein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as αB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of αB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of αB-crystallin against aggregation of the disease-related amyloid fibril forming protein α-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.Heath Ecroyd and John A. Carve
Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation
No full textThe capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.Mark A. Baker, Louise Hetherington, Heath Ecroyd, Shaun D. Roman, and R. John Aitke
Carboxymethylated-kappa-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation
No full textReduced and carboxymethylated-kappa-casein (RCM-kappa-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Abeta), which is associated with Alzheimer's disease. In this study, a series of flavonoids, many known to be inhibitors of Abeta fibril formation, were screened for their ability to inhibit RCM-kappa-CN fibrilisation, and the results were compared with literature data on Abeta inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-kappa-CN fibril formation. IC(50) values were between 10- and 200-fold higher with RCM-kappa-CN than literature results for Abeta fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-kappa-CN assay make it an economic alternative first screen for Abeta inhibitory activity, especially for use with large compound libraries.John A. Carver, Peter J. Duggan, Heath Ecroyd, Yanqin Liu, Adam G. Meyer, C. Elisabet Tranber
Endogenous redox activity in mouse spermatozoa and Its role in regulating the tyrosine phosphorylation events associated with sperm capacitation
No full text© 2003 by the Society for the Study of Reproduction, Inc.We investigated the role of endogenous redox activity in regulating the signal transduction pathway leading to tyrosine phosphorylation in mouse spermatozoa. Endogenous redox activity was monitored using a luminol-peroxidase chemiluminescent probe. Chemiluminescence increased in spermatozoa that were actively undergoing cAMP-mediated tyrosine phosphorylation events associated with capacitation and was inhibited in a dose-dependent manner by addition of catalase or diphenylene iodonium, both of which also inhibited tyrosine phosphorylation within the cell at points downstream of cAMP. Excluding bicarbonate from the incubation medium reduced the redox activity of sperm by 80–90% and dramatically reduced tyrosine phosphorylation. This study provides the first evidence that tyrosine phosphorylation associated with capacitation in mouse spermatozoa is redox regulated by a flavinoid-containing enzyme involving mediation by hydrogen peroxide. Bicarbonate regulated the redox activity of mouse spermatozoa, and this regulation may contribute to the impact of this anion on tyrosine phosphorylation during capacitation of mouse spermatozoa.Heath W. Ecroyd, Russell C. Jones, and R. John Aitke
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